title: A competitive chemical-proteomic platform to identify zinc-binding cysteines.
abstract: Zinc ions (Zn(2+)) play vital catalytic, structural, and regulatory roles in protein function and are commonly chelated to cysteine residues within the protein framework. Current methods to identify Zn(2+)-binding cysteines rely on computational studies based on known Zn(2+)-chelating motifs, as well as high-resolution structural data. These available approaches preclude the global identification of putative Zn(2+)-chelating cysteines, particularly on poorly characterized proteins in the proteome. Herein, we describe an experimental platform that identifies metal-binding cysteines on the basis of their reduced nucleophilicity upon treatment with metal ions. As validation of our platform, we utilize a peptide-based cysteine-reactive probe to show that the known Zn(2+)-chelating cysteine in sorbitol dehydrogenase (SORD) demonstrates an expected loss in nucleophilicity in the presence of Zn(2+) ions and a gain in nucleophilicity upon treatment with a Zn(2+) chelator. We also identified the active-site cysteine in glutathione S-transferase omega-1 (GSTO1) as a potential Zn(2+)-chelation site, albeit with lower metal affinity relative to SORD. Treatment of recombinant GSTO1 with Zn(2+) ions results in a dose-dependent decrease in GSTO1 activity. Furthermore, we apply a promiscuous cysteine-reactive probe to globally identify putative Zn(2+)-binding cysteines across ∼900 cysteines in the human proteome. This proteomic study identified several well-characterized Zn(2+)-binding proteins, as well as numerous uncharacterized proteins from functionally distinct classes. This platform is highly versatile and provides an experimental tool that complements existing computational and structural methods to identify metal-binding cysteine residues.
authors.affiliation: Department of Chemistry, Boston College , Chestnut Hill, Massachusetts 02467, United States.
authors.forename: Nicholas J
authors.initials: NJ
authors.lastname: Pace
authors.forename: Eranthie
authors.initials: E
authors.lastname: Weerapana
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D005982:Glutathione Transferase / Q000737:chemistry / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D007064:L-Iditol 2-Dehydrogenase / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D040901:Proteomics / Q000379:methods*; D015032:Zinc / Q000378:metabolism*
chemical_list: D007064:L-Iditol 2-Dehydrogenase; C490476:GSTO1 protein, human; D005982:Glutathione Transferase; D015032:Zinc; D003545:Cysteine